1. Field of the Invention
This invention relates to a depilling treatment of unfinished cotton fabric pieces, or finished garments, with cellulase enzymes, wherein the treatment removes less than approximately 3.0% of the initial fabric weight. More specifically, this invention relates to a method for reducing or preventing fabric strength loss during depilling by employing certain specific cellulase mixtures, and preferably Trichoderma cellulase enzyme mixtures consisting essentially of at least 80% EGII. It has been discovered that with enzyme mixtures enriched in EGII, the removal of pills is more efficient, and fabric destruction during enzyme treatment can reduced by about 88%, relative to the standard commercial cellulase enzymes now in use.
2. Brief Description of the Prior Art
Cellulase enzymes widely are used to improve the appearance and softness of cotton-containing fabrics and garments. As used herein, the terms "cotton goods" denotes finished or unfinished fabrics consisting of cotton or blends of cotton with other fibers.
One widespread application of cellulase enzymes is for treating cotton-containing fabrics so as to "stonewash" denim, in which cellulase enzymes largely have replaced stones for generating the soft, faded denim that is desired by consumers. Further details of cellulase for denim stonewashing can be found in Nielsen, et al, ENZYME APPLICATIONS (INDUSTRIAL), Encyclopedia of Chemical Technology, (Kirk-Othmer Publishers, 1993), vol. 9, p. 603-604 ( referred to hereafter as "Nielsen, et al").
A second widespread application of cellulase enzymes is for treating cotton-containing fabrics so as to remove cotton fuzz and loose surface fibers on or in the fabric, which categorically involves removal of less than approximately 3.0% of the initial fabric weight, and typically less than 1.0%. This process is known variously by the terms "depilling", "biopolishing", "biofinishing", and "reformation". The term "depilling" will be used herein to refer to all such fabric treatments. In depilling, the cellulase treatment smooths the surface of the fabric, which in turn imparts improved softness and appearance, thereby increasing the quality and value of the fabric. Cellulase treatment for depilling also helps to prevent the subsequent formation of fiber pills that make the garments appear worn, and improves the uniformity of the fabric by removing dead or immature cotton. Further details of cellulase for depilling further can be found in Nielsen, et al, at pages 595-604. Depilling of cotton-containing goods is the field of the present invention.
Shear stress is applied to cotton garments during garment manufacturing and in repeated wearing, washing, and tumble drying, and thereby damages the surface. A close look reveals the presence of fibrils ranging in size from a few microns to a few millimeters. The damaged surface scatters light, giving a dull, grayish appearance with decreased color brightness and contrast between different colors. Dust particles also tend to adhere to the damaged areas, adding to the gray appearance. The damaged fibers also make the surface more rigid, thereby decreasing hand, or softness.
Cellulase enzymes hydrolyze exposed beta-1,4 bonds in cellulose. This leads to removal of the fibrils, which are the most exposed part of the fabric. The removal of fibrils is believed to directly improve the softness of the garments and also to lead to better color and cleanliness, both by removing soil attached to the fibrils and by improving the penetration of other cleaning compounds being used. The removal of fibrils initially also helps to prevent a subsequent formation of fibrils.
Cellulase enzymes have several advantages over conventional fabric softeners used to improve the smoothness and sheen on cotton fabrics. Conventional softeners, which are primarily clay or cationic surfactants, coat the fabric and impart a greasy feel, which is undesirable. Softeners also decrease the water absorbency, which is a disadvantage for towels and the like. The enzymes are also preferred from an environmental point of view.
In a typical depilling treatment step during garment manufacturing, fabric (usually dyed), water, buffer, detergents, and enzyme are added to a rotating horizontal or vertical drum jet dyer, washing machine, or other device that provides agitation and shear to the fabric. The treatment is typically for 15 to 120 minutes at 35.degree. C. to 60.degree. C., at a pH of 4 to 6.5 . The ratio of liquor to fabric is usually between 2.5:1 and 6:1, by weight . The amount of cellulase enzyme added typically corresponds to a cellulase activity of about 1,000 to 200,000 CMC units per kilogram of fabric, based on the cellulase assay method of Ghose (1987). After treatment, the enzyme is often destroyed by heating the solution to 70.degree. C. for 10 minutes. The fabric is removed from the machine, dried, and prepared in rolls, sometimes after additional dying. A summary of publications that further describe details of conventional cellulase treatments for depilling of cotton fabrics during manufacturing is found in U.S. Pat. No. 5,232,851, at Column 1.
For a depilling treatment during a laundering step, the cellulase is included in a detergent mixture with the many other ingredients. The other ingredients might include other enzymes, such as proteases, lipases, and cellulases, as well as surfactants, buffers, builders, bleach, anti-redeposition agents, optical brighteners, anti-oxidants, and solubilizers.
One conventional detergent mixture containing cellulase enzymes is further described by Clarkson, et al, in U.S. Pat. No. 5,290,474, (hereafter referred to as "Clarkson '474"). The treatment is typically for 15 to 60 minutes at 20.degree. C. to 70.degree. C., and at a pH of 7 to 9.5. The ratio of liquor to fabric, by weight, is usually between 2.5:1 and 10:1. The amount of cellulase enzyme added typically corresponds to a cellulase activity of about 200 to 40,000 CMC units per kilogram of fabric, based on the cellulase assay method of Ghose (1987).
Cellulase enzymes are used for the depilling of cotton fabrics and of blends of cotton and man-made fibers, including lyocell, rayon, polyester, acrylic, nylon, and cellulose acetate. Further details are illustrated in Clarkson '474, at column 7. Cellulase treatment is carried out on fabrics or sewn garments comprising material made of cotton or cotton blends, with or without a resinous finish. Cellulase depilling may be carried out on fabrics of at least 40% cotton, by weight. However, results are more pronounced and economical if the cotton content is more than 60% by weight, and the best results are obtained if the cotton content is more than 75% by weight.
The cellulase enzymes from one particular genus of wood-rotting fungus, Trichoderma, often are used in depilling applications. Trichoderma cellulases are preferred in textile processing and laundering because of a highly potent action against cotton and other forms of cellulose. Trichoderma cellulase products are commercially available from Iogen Corporation of Ottawa, Ontario, Canada; Genencor International; Novo Nordisk; Enzyme Development Company, and others. Commercial cellulases such as Iogen Cellulase are referred to as "natural" or "complete" cellulases because they contain most, if not all, of the six most prevalent naturally occuring cellulase components: cellobiohydrolase I (CBHI); cellobiohydrolase II (CBHII); endoglucanase I (EGI); endoglucanase II (EGII); endoglucanase III (EGIII) and endoglucanase V (EGV).
The widespread use of complete cellulases for depilling attests to the usefulness of these enzymes. However, one disadvantage of such complete cellulases in depilling treatments is that they can cause a significant loss of strength of the fabric. See Clarkson, et al, U.S. Pat. No. 5,246,853, (hereafter "Clarkson '853"). Loss of strength arises from the action of the cellulase against cellulose on the main body of the fabric, rather than just the desired action against fuzz or pills. Excessive strength loss can cause damage to the fabric, such as pin holes or overly worn spots, and decrease the useful life of the fabric. Decreasing the strength loss would overcome these problems. In addition, decreasing the strength loss would allow one to achieve the desired appearance and softness in fabrics with higher strength than presently achievable. This would result in valuable new products for the industry and the consumer.
To decrease the loss of strength caused by Trichoderma cellulase, efforts have focused on the properties of the individual enzymes that comprise Trichoderma cellulase.
Trichoderma naturally makes a mixture of about two dozen different types of cellulase enzymes, which are individually known as components. Several of the most prevalent of these components have been identified and named, including cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII), endoglucanase I (EGI), endoglucanase II (EGII), endoglucanase III (EGIII), and endoglucanase V (EGV).
Each of the Trichoderma cellulase enzymes have been classified into an appropriate family of the more than 40 recognized families of hydrolase enzymes. Classification is based on the sequence of amino acids that comprise the enzymes and the three dimensional structure, as described by Claesssens and Henrissat, "SPECIFICITY MAPPING OF CELLULOLYTIC ENZYMES: CLASSIFICATION INTO FAMILIES OF STRUCTURALLY RELATED PROTEINS CONFIRMED BY BIOCHEMICAL ANALYSIS", in Protein Science vol. 1, p. 1293-1297 (1992). The approximate properties, classification, references for amino acid sequences, and proportion of total cellulase protein in the natural enzyme of various Trichoderma cellulase components are summarized, in TABLE 1.
TABLE 1 ______________________________________ Trichoderma Cellulase Components Enzyme Mol. wt. Isoelectric pt. Family Reference Conc. (%) ______________________________________ CBHI 63,000 4.3 7 A 50-60 CBHII 58,000 6.0 6 B 15-18 EGI 53,000 4.6 7 C 12-15 EGII 50,000 5.3 5 D 9-11 EGIII 25,000 7.4 12 E 0-3 EGV 23,000 3.7 45 F 0-3 ______________________________________ References: A. Shoemaker, et al, Molecular Cloning Of ExoCellobiohydrolase I Derived From Trichoderma Reesei Strain L27. BIO/TECHNOLOGY vol. 1, p. 691-696 (1983). B. Chen, et al, Nucleotide Sequence And Deduced Primary Structure Of Cellobiohydrolase II From Trichoderma Reesei. BIO/TECHNOLOGY, vol. 5, p. 274-278 (1987). C. Penttila, et al, Homology Between Cellulase Genes Of Trichoderma Reesei: Complete Nucleotide Sequence Of The Endoglucanase I Gene. GENE vol. 45, p. 253-263 (1986). D. Saloheimo, et al, EGIII, A New Endoglucanase From Trichoderma Reesei: The Characterization Of Both Gene And Enzyme. GENE, Vol. 63, p. 11-21 (1988). E. Ward, et al, U.S. Pat. No. 5,475,101 F. Saloheimo, et al, A Novel, Small Endoglucanase Gene, Egl5, From Trichoderma Reesei Isolated By Expression In Yeast. MOLECULAR MICROBIOLOG Vol. 61, p. 1090-1097.
It should be emphasized that the nomenclature used in TABLE 1 is that nomenclature as currently used in this field, and reflects certain changes from earlier nomenclature. For example, the component "EGII" incorrectly and widely had been referred to as--EGII--in early reference works. See, for example, the discussion in Stalbrand, et al, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 61, p.1090-1097 (1995) ).
One approach followed by prior art workers seeking to decrease the fabric strength loss due to depilling treatments has been to produce what is known as a "truncated version" of cellulase components.
Most cellulase components comprise a catalytic core domain and a cellulose binding domain, separated by a flexible linker that consists of several amino acids. Techniques have been reported for cleaving either the core domain or the binding domain. Techniques also have been reported for using Trichoderma strains with modified DNA, so as to encode only a desired portion of the cellulase. See the published patent document Fowler, et al, WO 95/16782,(hereafter "Fowler '782") The use of protease enzymes to cleave a desired portion of an enzyme also has been reported. See Woodward, et al, BIOTECHNOL. APPL. BIOCHEM., Vol. 19, p. 141-153 (1994).
These truncated cellulases have not solved the problems of strength loss in depilling, as reported by Kumar, et al in "OPTIMIZING THE USE OF CELLULASE ENZYMES IN FINISHING CELLULOSIC FABRICS", 1995 AATCC conference, Atlanta, page 238, (hereafter referred to as "Kumar, et al"). This paper compares performances in depilling (or "biofinishing" as the term appears therein) among Standard Whole Cellulase (which contains the six major cellulase components) and two novel cellulases. One novel cellulase is said to be a "modified acid cellulase", (i.e., a truncated cellulase prepared using procedures of Fowler '782), and does not show any decrease in fabric strength loss during depilling, relative to Standard Whole Cellulase.
A second approach towards decreasing fabric strength loss during depilling treatents has been to choose mixtures of cellulase components that offer advantages relative to the natural mixture. By using well known techniques of genetic engineering or protein processing (described in Clarkson '853), one can alter the relative amounts of cellulase components present.
Bjork et al, U.S. Pat. No. 5,120,463 (hereafter "Bjork '463") and the published patent document Clarkson, et al, WO 93/22428 (hereafter "Clarkson '428") teach that a cellulase enzyme enriched in CBHI components will have less strength loss, as well as superior performance in softening and improving the feel of cotton fabrics, than cellulase with its endoglucanases present. The enzyme mixture with best performance taught by Bjork '463 is 96% CBHI, 2% EGI, and 2% EGII (see TABLE 3 of EXAMPLE 3), with 500 ppm CBHI and 10 ppm of EGI and EGII, divided equally. This mixture also is indicated on FIG. 1 herein as "Bjork '463-best". Very poor performance is indicated for a mixture that is 45% EGI, 45% EGII, and 10% CBHI. This mixture, which is indicated as 10 ppm CBHI and 100 ppm divided between EGI and EGII, also is indicated on FIG. 1 herein as "Bjork '463-poor".
The teachings of Bjork '463 also are supported by the teachings of Cavaco-Paulo and Rios, "ANALYSIS OF THE MECHANICAL PROPERTIES OF CELLULASE TREATED FABRICS", (1996 AATCC conference, Nashville), at p. 129.
Clarkson, et al, U.S. Pat. No. 5,525,507 and Clarkson '853 both allege a particular cellulase enzyme composition for treating cotton fabric to achieve enhanced feel, softness, color enhancement and a stone washed appearance. That particular enzyme composition is taught necessarily to be substantially free of CBHI-type components (column 2, line 57). In one preferred embodiment, the enzyme also is taught necessarily to be free of CBHII components. In a second preferred embodiment, the enzyme is taught necessarily to have at least 10% endoglucanase components. In a third preferred embodiment, the enzyme is taught necessarily to have at least 20% endoglucanase components.
Clarkson '853 alleged best results with a mixture that was 50% EGI, 37% EGII, and 13% EGIII. That mixture is indicated on FIG. 1 herein as "Clarkson '853 best" and is described by Clarkson '853 as "CBHI and CBHII deleted" within Example 16. The necessary proportions of endoglucanases in that mixture as stated above were determined, as follows: In Clarkson '853, at Example 13, the proportions of cellulase components in the natural mixture are listed as being CBHI 45-55%; CBHII 13-15%; EGI 11-13%; EGII 8-11%; EGIII 1-4%. At Example 16, the preferred enzyme is said to have all CBHI and CBHII deleted. If CBHI and CBHII are removed from the total mixture, and the average concentrations of the remaining enzymes are normalized to total 100%, the result will be as stated above.
The second best performance shown by Clarkson '853 was a mixture that was 37% CBHII, 32% EGI, 24% EGII, and 7% EGIII. This mixture is indicated on FIG. 1 as "Clarkson '853 2nd best" and is described by Clarkson '853 as "CBHI deleted". The proportions of enzymes in this mixture as stated above were determined, as follows: In Clarkson '853, at Example 13, the proportions of cellulase components in the natural mixture are listed as being CBHI 45-55%; CBHII 13-15%; EGI 11-13%; EGII 8-11%; EGIII 1-4%. At Example 16, the second-best enzyme is said to have all CBHI deleted. If CBHI is removed from the total mixture, and the average concentrations of the remaining enzymes are normalized to total 100%, the result will be as stated above.
The third best performance shown by Clarkson '853 was a mixture that was 68% CBHI, 16% EGI, 12% EGII, and 4% EGIII. This mixture is indicated on FIG. 1 as "Clarkson '853 3rd best" and is described by Clarkson '853 as "CBHII deleted". The proportions of enzymes in this mixture as stated above were determined, as follows: In Clarkson '853, at Example 13, the proportions of cellulase components in the natural mixture are listed as being CBHI 45-55%; CBHII 13-15%; EGI 11-13%; EGII 8-11%; EGIII 1-4%. At Example 16, the third-best enzyme is said to have all CBHII deleted. If CBHII is removed from the total mixture, and the average concentrations of the remaining enzymes are normalized to total 100%, the result will be as stated above.
The worst performance reported by Clarkson '853 was with the natural cellulase mixture, which also is indicated on FIG. 1 herein.
Clarkson et al, U.S. Pat. No. 5,290,474 (hereafter "Clarkson '474") claim the use of enzymes containing at least 40% EGIII for treating cotton. In a preferred embodiment, the enzyme consists of no more than 5% CBHI components, and at least 70% EGIII. Clarkson '474 claim that this mixture of enzymes is advantageous in that it can be used in alkaline pH (column 3, line 58). There is no suggestion that these mixtures of enzymes result in less strength loss than the mixtures taught by Clarkson '853.
The single example of cellulase mixtures described by Clarkson '474 is substantially pure EGIII. This is indicated on FIG. 1.
The patent publication, Saloheimo, et al, WO 94/28117 relates to uses for the component EGV endoglucanase. This enzyme is taught to be active at alkaline pH and is recommended for use in the textiles industry (page 16, line 19). However, Saloheimo et al neither disclose nor suggest if this enzyme might be superior in depilling performance to other mixtures or components described in the prior art.
Kumar, et al describe measured performances in depilling with Standard Whole Cellulase which contains all of the major cellulase components, and also for two alleged "novel cellulases". One of the novel cellulases so denoted is said to be an "enriched endo-cellulase" but the components present or removed are not identified. Kumar, et al allege that the "enriched endo-cellulase" enzyme causes less strength loss in depilling than would Standard Whole Cellulase. This "enriched endo-cellulase" was not, however, recommended for applications with high abrasion requirements such as heavy cotton and lyocell, since high doses and additional time are said to be required (page 243, middle paragraph).